Diarrheal diseases are a significant global problem resulting in high levels of morbidity and mortality especially to children under the age of 5. Two of the most prominent agents that cause diarrheal disease are Shigella spp and enterotoxigenic E. coli. (ETEC). Current measures for preventing and treating these diseases are insufficient in that over 375 million cases occur with an estimated 1.48 million resulting in death annually (34).
Shigella spp are invasive pathogens that can cause disease through ingestion of food or water contaminated with as little as 100 bacteria. Shigella can penetrate the intestinal epithelial cells of the colonic mucosa and stimulate a mucosal inflammatory response eliciting the production of an array of proinflammatory cytokines leading to recruitment of neutrophils and macrophages (reviewed in (15)). Following invasion, the bacteria multiply and spread to contiguous cells using actin polymerization (25). The resulting disease shigellosis (bacillary dysentery) is characterized by an inflammatory condition of the colon with accompanying fever, vomiting, severe abdominal pain, diarrhea and passage of blood and mucus-containing stools (13).
Enterotoxigenic E. coli (ETEC) is also transmitted through contaminated food or water, however the infectious dose for ETEC is much higher (11). Once ingested, ETEC attach to mucosal epithelial cells using proteinacious fimbriae or colonization factor antigens (CFAs or CFs) and can secrete up to two separate enterotoxins designated heat-stable toxin (ST) and heat-labile toxin (HLT) (reviewed in (19) and refs therein). Immune response to ETEC infections indicates secretory IgA (sIgA) directed towards CFs can provide protective immunity against homologous fimbrial type (11).
Currently, there is a significant effort being put forth toward the development of a safe and efficacious vaccine for both of these enteric diseases. These efforts include: subcellular complexes purified from virulent Shigella (30), detoxified LPS-conjugates, subunit approaches, killed whole-cell preparations, and attenuation of pathogenic isolates for use as live attenuated vaccines (34).
Precise or targeted attenuation of pathogenic (invasive) strains of Shigella has made significant progress over the past 15 years. Initially attenuating mutations were made in key biosynthetic pathways creating auxotrophic mutants, which maintain the invasive nature of Shigella. These mutations tended to reduce or eliminate intracellular replication once inside the host cytoplasm. However, a greater understanding of the molecular pathogenesis of Shigella has led to the targeting of specific virulence factors (24) and reviewed in (15). One such strain, SC602 has deletions in both IcsA and IucA (6). This strain is highly invasive, however once inside host cells it cannot spread to contiguous cells due to the IcsA mutation. IcsA mutants, unlike wild-type strains, do not elicit a characteristic keratoconjunctivitis (Sereny reaction) when applied to the eyes of guinea pigs, SC602 has recently undergone phase 1 and 2 clinical trials in North American volunteers and demonstrated significant protection against severe shigellosis (7, 20). However, the vaccine can be reactogenic at doses higher than 104 thus demonstrating the need to balance attenuation with immunogenicity.
In addition to their potential for protection against shigellosis, attenuated strains of Shigella have been used as delivery vehicles for genes encoding numerous other protective antigens (1, 3-6, 21, 23). In one scenario the heterologous genes are regulated using a prokaryotic promoter and expressed by the attenuated bacteria. Immunogenicity of the antigen in this situation depends on the subcellular location and of the antigen within the bacteria (17, 22). The heterologous antigen is then processed by the immune system along with other bacterially derived antigens. Alternatively, the bacteria harbor heterologous genes under the control of a eukaryotic promoter. These so-called DNA vaccines are delivered to antigen presenting cell (APCs) following invasion and bacterial lysis. Once inside the APC the eukaryotic promoter is turned on and the expression of foreign proteins leads to an immune response (reviewed in (32)). Regarding the former scenario several considerations must be considered when expressing pathogen-derived heterologous protein antigens in attenuated bacterial vectors regardless of the antigen and species of bacteria. First, the antigen must be expressed at optimum level so as to minimize further attenuation of the vaccine strain. The second consideration is the cellular location and thus presentation to the immune system. A comparative study looking at antigen subcellular location (periplasmic or secreted vs. cytoplasmic) found that periplasmic and extracellular antigens are more immunogenic that antigens retained in the cytoplasm (17). Finally, the goal is to create a multivalent vaccine strain and thus heterologous antigen expression should not reduce the immunogenicity (invasiveness) of the bacterial vector.
There are several reports of Shigella being used as a carrier of both heterologous protein and DNA antigens. In particular, the Center for Vaccine Development (CVD) of the University of Maryland School of Medicine has set out and made significant progress towards the goal of constructing a combination vaccine to protect against Shigella and ETEC-associated diarrhea (1, 2, 4, 21, 23). They have used the engineered Shigella vaccine strain CVD 1204 (ΔguaBA) to express several ETEC fimbrial antigens as well as mutant heat-labile toxin (mLT) (1, 2, 4, 21). The CVD's approach has been to clone and express the entire fimbrial operon under the control of an inducible promoter. To date they have constructed and tested Shigella strains that express CFA/I, CS2, CS3, CS4, as well as detoxified mLT (LThK63 or LThR72). Guinea pigs immunized with mixed inoculums containing five different Shigella strains, each expressing individual ETEC fimbriae, showed serum and mucosal antibody responses to both the Shigella vector and the ETEC fimbriae (4).